CD11b+ cells were isolated from mouse xenograft tumors or Matrigel plugs by magnetic sorting using CD11b MicroBeads (Miltenyi Biotec GmbH, Bergisch-Gladbach, Germany). In brief, tissues minced in PBS were incubated in collagenase L (Nitta Gelatin, Osaka, Japan) and DNase I (Roche Diagnostics GmbH, Mannheim, Germany) at final concentrations of 0.5% and 20 unit/mL. The mixture was incubated for 1 h at 37°C under gentle agitation. Digestion was stopped using fetal bovine serum, after which the cell suspension was washed and then passed through a 70-µm-mesh nylon screen. The cells were incubated with CD11b MicroBeads for 15 min at 4°C and loaded onto a MIDIMACS column (Miltenyi Biotec GmbH) according to the manufacturer's instructions. Isolated CD11b+ cells from xenografts or Matrigel were used as TAMs for further experiments. For cell surface staining, single-cell suspensions were incubated with fluorescein isothiocyanate–conjugated anti-CD11b monoclonal antibody (BD Bioscience) and phycoerythrin-conjugated anti-F4/80 monoclonal antibody (eBioscience, Inc., San Diego, CA) for 15 min at 4°C. The stained cells were run on a FACSCalibur flow cytometer (BD Biosciences). The data were analyzed using the CellQuest software program (BD Biosciences).
Midimacs column
Manufactured by Miltenyi Biotec
Sourced in Germany
The MIDIMACS® column is a magnetic separation column designed for the efficient isolation and purification of cells, proteins, or other biological molecules. The column utilizes a magnetic field to capture the target molecules or cells, allowing for the separation and collection of the desired components from a complex sample.
Automatically generated - may contain errors
Lab products found in correlation
Facscalibur flow cytometer, by BD (1 mentions)
Cd11b microbead, by Miltenyi Biotec (1 mentions)
Dnase 1, by Roche (1 mentions)
Cellquest software program, by BD (1 mentions)
Collagenase l, by Nitta Gelatin (1 mentions)
Fitc conjugated anti cd44 antibody, by Thermo Fisher Scientific (1 mentions)
Macs cell separation system, by Miltenyi Biotec (1 mentions)
Edta vacutubes, by BD (1 mentions)
Ficoll paque plus protocol, by GE Healthcare (1 mentions)
Cd56 magnetic beads, by Miltenyi Biotec (1 mentions)
Automacs, by Miltenyi Biotec (1 mentions)
Cd8a t cell isolation kit, by Miltenyi Biotec (1 mentions)
6 protocols using midimacs column
1
Isolation and Characterization of Tumor-Associated Macrophages
2
Isolation of ZIKV-infected Müller Cells
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Müller cells were isolated using the MACS cell separation system (Miltenyi Biotec, San Diego, CA, USA). ZIKVFSS-infected retina tissues were harvested at 6 days post infection (dpi) and digested with collagenase. The cell suspension was first labeled with FITC-conjugated anti-CD44 antibody (eBioscience, San Diego, CA, USA), followed by binding to magnetic bead-conjugated anti-FITC antibody. Tagged cells were isolated with a MidiMACS column (Miltenyi Biotec, San Diego, CA, USA). Both Müller cells and flow-through fractions were collected for downstream RT-PCR analyses.
3
Isolation of NK Cells from PBMC
4
Purification and Activation of Splenic DCs and CD8 T Cells
5
Isolation and Purification of T and B Cells
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Peripheral blood mononuclear cells (PBMC) were isolated from heparinized blood as described [30 (link)]. CLL B cells from PBMC were depleted by filtration through a nylon wool column (Biotest, Breiech, Germany) [31 (link)] and T cells were further enriched by immunomagnetic depletion of B cells, NK cells and monocytes using MidiMACS columns and anti-CD19, anti-CD56 and anti-CD14 MACS MicroBeads (MiltenyiBiotec, Bergisch Gladbach, Germany) according to the manufacturer's recommendations. The purity of CD3 T-cells was 93–99% as determined by flow cytometry. CD4+ and CD8+ T cell were further purified from CD3+ T cells using the MACS MicroBeads negative selection kit (MiltenyiBiotec, Bergisch Gladbach, Germany). The purity of CD4+ and CD8+ cells were 96% and 94%, respectively. The purity of B cells (CD19+) was >99% as determined by flow cytometry.
6
Isolation of Myeloid-Derived Suppressor Cells
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After mechanical and enzymatic dissociation of induced TiRP tumors, myeloid-derived suppressor cells (MDSCs) (as a mix of monocytic and granulocytic cells) were purified by magnetic sorting. Briefly, CD11b+ cells were isolated using magnetic microbeads conjugated with monoclonal rat anti-mouse CD11b antibody. Depending on the experiment, MDSC isolation was carried out by either biotinylated Ly6G and Gr-1 mAbs together or alone, with anti-biotin-coated or streptavidin-coated microbeads. All separations were performed using Miltenyi Biotec kits and MidiMacs columns. Purity of cell populations was evaluated by flow cytometry.
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